Project Summary Information in the nervous system is relayed mostly at synapses, where neurotransmitter is released with great temporal precision from a presynaptic terminal on to a post-synaptic cell via the fusion of membrane bound synaptic vesicles (SVs) with the cell membrane, in a process called exocytosis. The components of these SVs are subsequently retrieved via endocytosis and recycled for reuse. This grant aims to understand the interplay between SV recycling and membrane tension gradients and associated membrane flows. In neurons and neuroendocrine cells, both exocytosis and endocytosis are influenced by osmotic swelling or shrinking, suggesting they are influenced by membrane tension, ?. Conversely, membrane addition to the presynaptic terminal via exocytosis is expected to lower ?, while endocytosis should restore it. In addition, membrane tension has been suggested to be one of the possible signals for coupling exocytosis to endocytosis. However, despite these key roles, there are no measurements of membrane tension in synaptic terminals and how tension changes are related to exo-endocytosis is not known, mainly due to technical difficulties. The best method to probe ? is to pull a thin membrane tether from the cell surface using optical tweezers, manipulating a 1-3 ?m diameter bead as a handle. The bead's displacement from the trap center provides the tether force, which reflects ?. However, most terminals are small and are tightly coupled to post-synaptic structures, making tether pulling impractical. We overcome this challenge using goldfish bipolar cells which possess giant terminals, in a setup that combines optical tweezers with electrophysiology (to control stimulation and/or measure capacitance changes) and with high-resolution fluorescence microscopy (to label and identify sub- cellular structures and calcium imaging). We aim 1) to characterize the tether force response to electrical and mechanical perturbations that occur at a presynaptic terminal during activity. After stimulation, membrane added at an exocytic site needs to flow (and the associated tension perturbation propagate) over the terminal surface, then through the tether to produce a change in the measured tether force. We will characterize membrane flows in double-tether experiments and calibrate the tether response to step- changes in tether length. We will confirm that ? changes we observed in preliminary experiments (a drop ~1 s after stimulation, followed by recovery in ~10 s) are due to exo-endocytosis, and characterize rapid voltage- induced tether force changes. These will enable a quantitative understanding of measured ? changes associated with stimulation. Next, we will 2) characterize how membrane tension is regulated at a presynaptic nerve terminal. Combining pharmacological interventions with live imaging and ? measurements, we will test the hypothesis that F-actin is a major regulator of ? at the nerve terminal. We will manipulate ? and calcium independently to dissect calcium and ? requirements for SV turnover. These measurements will help generate a model of feedback between membrane trafficking and ? at the nerve terminal.